1. Field of the Invention
The present invention relates to a process and a device for the parallel preparation of at least 4n oligonucleotides.
2. Description of the Related Art
DE 42 06 488 A1 discloses a process and a device for the preparation of oligonucleotides. The known device has four bars one above the other, the contact surfaces of which are worked by grinding and polishing such that the bars can be displaced relative to one another without a gap. One of the bars contains reactions spaces which can be filled and emptied via entry and exit lines in the other bars. The individual reaction spaces are filled successively with reagents. In order for the said contact surfaces of the displaceable bars to be sealed well with respect to one another, very precise working of these contact surfaces is necessary and the bars must be made of wear-resistant material, for example of stainless steel or of particular glass materials.
The demand for oligonucleotides is increasing constantly and there is therefore the desire to prepare the highest possible number of oligonucleotides inexpensively, in a short time and with a high quality. The oligonucleotides here can be the same or different.
The invention is therefore based on the object of providing an improved process and an improved device for the preparation of oligonucleotides which takes into account the abovementioned desire.
To this end the invention provides a process for the parallel preparation of at least 4n oligonucleotides. In the process according to the invention, at least four inserts each with n reaction vessels (n≧1) are arranged on or in a plate such that a first insert is at a first station, a second insert is at a second station, a third insert is a third station, and a fourth insert fourth station. Each reaction vessel contains an initiator base which is necessary for the synthesis of oligonucleotides and is bound, for example, to an inert carrier. Instead of an initiator base, it is also possible to use a so-called universal carrier known to experts. Porous glass, so-called controlled pore glass (CPG), can be used e.g. as the carrier material. A series of operations for oligonucleotide synthesis is then carried out in parallel at the four stations mentioned.
Thus, a so-called deblocking operation, by means of which protective groups present on the initiator bases are split off, so that individual nucleotide units can be coupled to the initiator base later, is carried out simultaneously in all n reactions vessels of the insert at the first station. These protective groups are also called DMT groups. Simultaneously with the deblocking operation taking place at the first station, a first washing operation by means of which the protective groups split off beforehand are washed out of the reaction vessels takes place at the second station, again simultaneously in all n reaction vessels at the second station. Also simultaneously with the two abovementioned operations, a so-called coupling operation by means of which the desired individual nucleotides are coupled on to the initiator bases or nucleotide chains in the reaction vessels takes place in all n reaction vessels at the third station. Again at the same time as the abovementioned three operations, a sequence of operations takes place simultaneously in all n reaction vessels of the insert at the fourth station, namely first a second washing operation, followed by a so-called capping operation, by means of which those oligonucleotides which have not undergone the desired chain lengthening in the preceding coupling operation are blocked in the reaction vessels, followed by a third washing operation, followed by an oxidation operation for stabilizing the phosphate foundation matrix of the oligonucleotides, and finally a fourth washing operation.
According to the invention, either the rotary plate is rotated station by station through the four stations mentioned, so that each insert passes through the individual stations in succession, or the stations are moved further by in each case one station relative to the inserts. This relative movement station by station between the inserts and the stations takes place until the desired oligonucleotides have been formed by coupling of the individual nucleotides to one another.
The synthesis cycle realized according to the invention by means of passing through the four stations is known per se and therefore does not need to be explained in detail. Nevertheless, by means of the process according to the invention this synthesis cycle is worked through in an extremely time-saving manner, in that firstly all n reaction vessels at a station are subjected simultaneously to the operation taking place at this station or to the operations taking place there, and in that secondly operations take place in parallel at all four stations. Furthermore, the process according to the invention utilizes in an intelligent manner the circumstance that the deblocking operation carried out at the first station and the coupling operation carried out at the third station are the operations which take the longest (and therefore determine the residence time required per station), by carrying out the significantly faster capping and oxidation operations successively at only one station (the fourth station).
The two measures mean that the process according to the invention is considerably faster compared with known processes for oligonucleotide synthesis. The productivity is increased in this manner, and the production costs for oligonucleotides can be lowered. Moreover, larger amounts of oligonucleotides can be provided in a shorter time. For example, if 24 reaction vessels are present per insert and if a period of time of 60 seconds is estimated as the cycle time per station, 96 oligonucleotides each with 20 nucleotide units can be produced within approx. 90 minutes. Assuming furthermore that a new run can be started about every 100 minutes, a production of 1,350 oligonucleotides per day is achieved with the process according to the invention. This corresponds to virtually 40 times the amount which can be prepared with the apparatus according to the abovementioned DE 42 06 488 A1.